PHF2 regulates sarcomeric gene transcription in myogenesis

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification.


Introduction
Skeletal muscles are responsible for basic functions such as locomotion and metabolism [1].Skeletal muscles are formed via myogenesis during development.Myogenic Regulatory Factors (MRFs, namely Myf5, Myod, Mrf4, and Myog) are the basic loop helix-type transcription factors that regulate each step of the myogenesis [2][3][4].Myogenesis, which involves coordination between cell proliferation and differentiation, participates in complex gene expression regulatory networks that exert their functions primarily by regulating intercellular signaling and the expression of specific genes.Differentiated myotubes express high levels of sarcomeric genes such as members of the myosin heavy chains (MyHC) family.Myogenic gene expression is regulated by multiple factors including DNA methylation, histone modifications, chromatin remodeling, and non-coding RNAs [5].
To address this, we evaluated the function of Phf2 in C2C12 myoblast myogenesis, hypothesizing that PHF2 participates in myogenesis by demethylating H3K9me2.We generated Phf2 knockout (KO) C2C12 myoblasts using the CRISPR/Cas9 system, and evaluated Phf2 function in myogenesis by evaluating RNA-sequencing-based global transcriptional changes.The loss of PHF2 in C2C12 myoblasts reduced the expression of genes that compose muscle sarcomere structure.These findings provide evidence for an important function of PHF2 in regulating gene expression.

Generation of Phf2 KO C2C12 myoblasts
Transfection of pGuide-it plasmids was performed using jetPRIME reagent (101000027, Polyplus, Illkirch-Graffenstaden, France), according to the manufacturer's instructions.Briefly, C2C12 cells were seeded in DMEM with 10% FBS at 5 × 10 4 cells/well in 6-well plates on the day before transfection.Four pGuide-it-ZsGreen plasmids (500 ng each) were pooled and transfected into the C2C12 cells; 24 h after transfection, cells expressing ZsGreen1 were sorted using a cell sorter (SH800, Sony, Tokyo, Japan).Sorted cells were subcultured for expansion for six days.Then the cells were stored at -80˚C.

RNA extraction
Cryopreserved Phf2 KO and mock cells were differentiated into myotubes, and total RNAs were extracted at 0, 2, 4, and 7 days post differentiation using ISOGEN Reagent according to the manufacturer's instructions and was quantified using a Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific).RNA purity was assessed using the 260 nm/280 nm ratio.

Quantitative Real-Time PCR Analysis (qRT-PCR)
Reverse transcription of total RNA (500 ng) was performed using the PrimeScript RT Master Mix (RR036A, Takara).qRT-PCR was performed using a final volume of 10 μl with Power SYBR Green PCR Master Mix (4367659, Thermo Fisher Scientific) according to the manufacturer's instructions.qRT-PCR was conducted using the StepOne Real-Time PCR System (Applied Biosystems, Waltham, MA, USA).The primer sequences are listed in Table 1.Actb was used as an internal control.

RNA-sequencing
Total RNAs extracted from the C2C12 cells on day 2 after differentiation were used for RNAseq.Libraries were prepared using the NEBNext Poly(A) mRNA Magnetic Isolation Module (for PolyA selection) (E7490, New England Biolabs, Ipswich, MA, USA) and total RNA using the NEBNext Ultra ll Directional RNA Library Prep Kit (E7760, New England Biolabs) according to the manufacturer's instructions.Sequencing reads were performed using a NovaSeq 6000 device (Illumina, San Diego, CA, USA) with a NovaSeq 6000 S4 Reagent Kit v1.5 (Illumina) using paired-end reads of 150 bp.
The reads were aligned against the mm10 mouse genome using HISTAT2, and the count of reads per gene was obtained using StringTie.Statistical analysis of differentially expressed genes was performed using exactTest in edgeR after normalization.RNA-seq data were deposited in the Gene Expression Omnibus (accession GSE226618).Gene enrichment, KEGG pathway, and BP DIRECT analyses were performed using DAVID Bioinformatics Resources [19,20].Principal component analysis (PCA) and hierarchically clustered heatmaps were analyzed using iDEP.96[21] (http://bioinformatics.sdstate.edu/idep/).

CUT&RUN-qPCR analysis
CUT&RUN-qPCR was performed using the CUT&RUN Assay Kit (86652, CST) according to the manufacturer's instructions.The Phf2 KO and mock cells 4 d post differentiation were subjected for CUT&RUN-qPCR analysis.The 100,000 cells were harvested per reaction and bound to activated Concanavalin A beads and permeabilized.The bead-cell complex was incubated overnight with either an anti-Histone H3K9me2 antibody (ab1220, Abcam, Cambridge, UK) or a control IgG antibody (CST) at 4˚C.Cells were washed three times with digitonin buffer, and resuspended in 50 μl pAG/MNase for 1h at RT.DNA fragments were purified using the NucleoSpin Gel and PCR Clean-up (740609.50,Takara).
Quantitative PCR was performed with GeneAce SYBR qPCR Mix II (313-09423, NIPPON GENE, Tokyo, Japan).The primer sequences are listed in Table 2.Each Ct values were normalized to Actb.

Statistical analysis
Results are expressed as mean ± SD.Statistical comparisons between two groups were performed using unpaired Student's t-tests.One-way ANOVA was used to analyze differences between multiple groups.

PHF2 expression in C2C12 cells
We examined Phf2 expression in C2C12 myotubes.Phf2 mRNA expression during C2C12 myotube differentiation was determined (Fig 1A).Phf2 was detected in C2C12 cells and its expression remained constant during differentiation.PHF2, a nuclear protein, is expressed in various organs [16,22].Therefore, PHF2 localization was visualized via immunofluorescence.PHF2 was localized to the nuclei of C2C12 myoblasts and myotubes (Fig 1B).This PHF2 localization is consistent with its function as an H3K9me2 histone demethylase.

Generation of Phf2 KO C2C12 cells via CRISPR/Cas9
To clarify the function of Phf2 in myogenesis, we generated Phf2 KO C2C12 myoblasts.We designed four distinct Phf2-specific gRNA sequences using CHOPCHOP and constructed four plasmids containing the ZsGreen fluorescence reporter [18].The four plasmids were pooled and transfected into C2C12 cells.As mock control, C2C12 cells transfected with a plasmid containing the ZsGreen fluorescence reporter without a specific gRNA was used.24 hours after transfection, the cells were examined via fluorescence microscopy to confirm that the plasmid DNA was incorporated into the C2C12 myoblasts.Transfected cells were collected using a cell sorter (Fig 2A and 2B).PHF2 protein levels, determined using the Simple Western System, were 96.5% lower following Phf2 knockout (Fig 2C ).

Differentiation of Phf2 KO C2C12 cells
To analyze the myotube-formation capacity of Phf2 KO cells, they were cultured for 7 d in the differentiation medium (Fig 3A

PHF2 regulates the expression of genes involved in muscle terminal differentiation via demethylating repressive H3K9me2 mark
We validated the RNA-seq results via qPCR and determined the expression patterns of the main genes in myogenesis.Myod1, Myf5, and Myog are myogenic regulators of transcription factors that induce myotube formation [24][25][26].qRT-PCR revealed that Myod1 and Myog were upregulated in the mock cells 2 d post differentiation, and that Myod1, Myf5, and Myog  Then we analyzed the expressions of terminal differentiation genes (Mef2c, Myhs, and Tnnt2) that are highly expressed in differentiated myotube and mainly encode muscle sarcomeres.We also included Mylk2 and Mybpc2 which were not highly expressed at 48 h post differentiation but were highly elevated on 7 d post differentiation.Their induction was significantly impaired in Phf2 KO myotubes (Fig 5A).These results indicated that Phf2 regulated the expression of sarcomeric genes highly expressed at terminal differentiation.
As PHF2 is known to act as an epigenetic activator by removing H3K9me2 [22,27,28], we performed CUT&RUN-qPCR assay with a H3K9me2 antibody using Phf2 KO and mock cells on

Discussion
In this study, we generated Phf2 KO cells using the CRISPR/Cas9 system and analyzed their transcriptional function during myogenesis via RNA sequencing.This revealed that PHF2 is required for the expression of sarcomeric genes such as Myhs and Mybpc2.
The CRISPR/Cas9 system was used to generate KO cells for the genes of interest [29][30][31].Using the CRISPR/Cas9 system to obtain KO cells, single-cell cloning is required, owing to the heterogeneity of mutations.However, repeated passage can easily result in the loss of myogenic function in C2C12 cells.Therefore, single-cell cloning was not performed after sorting.Instead, to increase the knockout efficiency, we developed four distinct gRNAs and transfected the pooled plasmid (Fig 2).The Phf2 KO cells remained viable even after cryopreservation.The KO cell generation method used here could be useful for the KO of other cells with differentiation capacities, such as 3T3-L1 or MC3T3-E1 cells.
These findings reveal that Phf2 KO impaired the expression of genes involved in myogenesis (Myog and Myod1) and in sarcomeric processes (Myhs and Mybpc2) (Figs 4 and 5).However, myotube formation (in terms of myotube diameter, total myotube area, and fusion index) did not differ significantly between Phf2 KO and mock cells (Fig 3).Conversely, suppression of sarcomeric genes may affect myofunctions such as muscle contraction, energy metabolism, and fiber-type changes.Maximal fast-twitch muscle strength is indeed suppressed in Mybpc2 mutant mice [32].
Additionally, it remains unclear whether these genes are direct targets of PHF2.To answer these questions, future ChIP-seq experiments and physiological analyses of muscle function in PHF2 deficient mice are necessary.Systemic Phf2 KO mice exhibit approximately 70% mortality within 3 d of birth, with reduced adipose tissue mass in the surviving mice [14].The generation of skeletal muscle-specific Phf2 KO mice is essential to further elucidate the function of PHF2 in skeletal muscles.

Limitations of the study
In this study, we used the mock control as a control of Phf2 KO cells.The mock cells were introduced the control plasmid containing Cas9, ZsGreen, and gRNA.However, the gRNA sequence of the control plasmid does not match the mouse genomic sequence.Therefore, the double-strand break would not happen in the mock control cells.The effect of double-strand break itself on C2C12 transcription is not excluded in this study.In addition, C2C12 cells produce immature myotubes compared to skeletal muscle in vivo, therefore, the present study could not clarify the role of Phf2 in sarcomere formation and muscle contractility.

Fig 1 .
Fig 1. Phf2 expression and PHF2 localization in C2C12 myotubes.(A) Phf2 mRNA expression during differentiation in C2C12 myotubes (n = 4 wells per condition in one experiment).(B) Immunofluorescence staining of PHF2 (green) and DAPI (blue) in C2C12 myotubes on days 0 and 7 post differentiation.Scale bar, 100 μm.https://doi.org/10.1371/journal.pone.0301690.g001 expression was slightly reduced in Phf2 KO cells, however, the expression levels were not changed at 7 d post differentiation (S1 Fig).Considering the results of myotube formation (Fig 3), the reduction of Myod1 and Myog at 2 d post differentiation is not physiologically important in early myogenesis.

Fig 2 .
Fig 2. Generation of Phf2 knockout (KO) cells in C2C12 myoblasts.(A) Overview of the generation of Phf2 KO C2C12 cells.(B) Myoblasts expressing ZsGreen were obtained via cell sorting.Sorted untransfected C2C12 cells (without fluorescence) were used as negative controls.Phf2 KO and mock cells were sorted under the same conditions.Sorted cells are indicated in the orange areas.(C) Protein levels of PHF2 were determined using the Simple Western System.https://doi.org/10.1371/journal.pone.0301690.g002 4 d post differentiation.Phf2 KO myotubes increased the H3K9me2 mark at the promoter region of Mef2c, Mybpc2, and Myh7 (Fig 5B).Taken together, these findings indicate that

Fig 4 .
Fig 4. RNA-sequence analysis of Phf2 knockout (KO) cells at 2 d post differentiation.(A) Principal components analysis with RNA-seq analysis of Phf2 control cells and Phf2 KO cells (n = 3 wells in each group in one experiment).(B) Volcano plot of the RNA-seq data obtained from Phf2 KO cells 2 d post differentiation.(C) Heatmap visualizing the expression profiles based on RNA-seq analysis of Phf2 control and Phf2 KO cells (n = 3 wells per group).(D) KEGG pathway analysis of differentially expressed genes (DEGs) in Phf2 KO C2C12 cells.Numbers in parentheses indicate the number of genes

Table 2 . The primer sequence for qPCR.
).The myotube diameter in the Phf2 KO group after 7 d differentiation was not significantly different from that of the mock group (Fig3B).The differentiation status of Phf2 KO cells 7 d after induction of differentiation was analyzed by immunofluorescence staining of MyHC (Fig3C).Phf2 KO did not affect total myotube area or myotube fusion index (Fig 3Dand 3E).These results suggest that Phf2 KO did not affect C2C12 myotube formation.